In recent years, from the original detection by clinical laboratory samples to the rapid detection realized in the doctor's office or point of care test, the field of medical health detection has been changed dramatically. A disposable biosensor with electrode system has been frequently used in rapid detection. Taking daily glucose detection of a diabetic as an example, nowadays, the glucose level of a diabetic can be immediately known by using a disposable biosensor at the diabetic's home, and it will help the diabetic to know his/her own physical condition, thereby adjusting his/her dietary structure, or going to the hospital and asking the doctor to adjust his/her dosage of medication according to his/her daily monitoring data. Hence, the accuracy of this type of disposable biosensor is very important in the daily monitoring of diseases.
This type of biosensor with electrode system can be used in the detection of various kinds of substances. Depending on different analytes, reaction reagents added to the electrode system are also different. Taking the biosensor used for determining glucose level of blood as an example, at first, the reaction reagents such as glucose oxidase are added to a working electrode. When a blood sample is dropped to the working electrode, since the enzyme reaction reagent dissolves in blood, glucose in the blood will react with the oxidase on the working electrode, and then electron acceptor will be reduced. After the enzyme reaction finishes, the reduced electron acceptor will be electrochemically oxidized and the concentration of glucose in the sample can be obtained by calculating the current value of oxidation.
The basic structure of this type of biosensors includes an insulative substrate, on which there's an electrode system which at least contains a working electrode and a counter electrode, and at least one working electrode is covered by reaction reagents that produce detectable signals according to concentration of determined substance in the sample; and a channel for detecting sample is formed by separating layer, the channel is on the working electrode and the counter electrode. When the sample to be detected goes through the channel, it reacts with the reaction reagent in the channel thereby producing detectable signals. An instrument reads the testing results according to the signals.
The amount of reaction reagent in the channel for detecting the sample reaction has a great influence on the test result. Therefore, how to precisely control the amount of reaction reagents that acts in the reaction has been a big technical problem in this field. The amount of reaction reagents that acts in the reaction is equal to the product of area of sample channel and the thickness of reaction reagent layer, therefore, it is a major problem that shall be solved to accurately control both sizes of sample channels among different products produced at the same batch and the uniformity of thickness of reaction reagent.
To accurately control the sizes of sample channels can be obtained by improving the accuracy of moulds and selecting proper materials, however, it is a relatively difficult technical problem to control the uniformity of the thickness of the reaction reagents.
As shown in FIG. 1 of the U.S. Pat. No. 7,655,119, an electrode comprising an insulative substrate 16 that have a rectangle reagent-diffusion control zone 18, the electrode that needs reaction reagents and exposed by the rectangle reagent-diffusion control zone is rightly the active zone of the electrode. The reaction reagents have been dropped onto the control zone 18 and contact with the electrode, and other parts of the electrode that have not acted in the reaction will not be dropped by the reaction reagents since they are covered by the insulating plate 16, hence, the reaction reagents will be limited in the required area on the working electrode. We found that, the dropped reagents often can not fully cover the whole rectangle zone by dropping reaction reagents, for example, the reagents can not reach the four vertex angles of the rectangle reagent-diffusion control zone. It means that the products produced at the same batch have different reaction reagent-covered areas. Once the volumes of dropped reaction reagents are equal, the thicknesses of reaction reagents of the products produced at the same batch have deviations, so, the amount of the reaction reagents that act in the reaction would not be equal. The results obtained will be inaccurate by using the product for testing. When using this manner of dropping reaction reagents onto the electrodes, more serious undesirable phenomena occur. The reaction reagents can not fully cover the whole rectangle zone, and even part of reagents overflows the both sides of the rectangle reagent diffusion-control zone.
When testing the sample with the biosensor, usually, a very small amount of sample, for example, only 0.5 microliter to 1 microliter of liquid, is required. Biosensor has also been made into a very tiny structure, and the reaction reagents covered at the electrodes are even smaller. In the research of improving the accuracy of testing, people hardly notice that the diffusion of reaction reagent is various in such a small area of electrode and notice the effect on the testing results brought by the variety. People have always studied to improve the detection accuracy of biosensor from aspects of structure of electrode, material of electrode, testing methods and meters. However, we found that when applying rectangle reagent diffusion control zone to control the distribution of reaction reagents at the electrode, the area of rectangle control zone is very small. And the reagent will form a hemispheric drop in the groove of the rectangle control zone because of surface tension, which causes the uneven of reagent thickness at the electrode to form a result of thick center and thin edge, especially, at the edge of coatings area, it would be uneven, or the reaction reagents would concentrate on the center of electrode active region. The edge surface tension of hemispheric drop prevents the further diffusion around, which causes reagent blanks between reagent edge and four vertex angles, which means those four vertex angles can not be filled up with by reaction reagents. Some the active region of the electrode of biosensors produced in this way may be evenly filled up with by the reaction reagent, while some vertex angles in the active region of the electrode have no reaction reagents or the covered thickness are not uniform. The lack of reaction reagent at some parts of the electrode will cause that some samples fail in acting in the reaction, and the unevenness of thickness of the reaction reagents covered at the electrode will make the changes of reaction rate which does not conform the pre-established method, which will lead to the inaccuracy of testing results.
And just as in the China Patent 200480023924.8, method of adding reagent by insulating plate-covering has not been used, it uses seam coating to directly make reaction reagent coated on the electrode to create continuous strips. Because the reagent area of coating is very large, it even contains some parts that do not need the addition of reaction reagent. It will lead to the waste of reagent and increase product cost. On the other hand, due to the surface tension on the insulative substrate, the diffusion of the reagent has been suffered from certain resistance that hampers the even diffusion of reaction reagent in the electrode system, which makes the reaction reagent can not reach the pre-established sites.
Since the size of biosensor that is used to detect the glucose concentration of human's blood is very tiny, and the detection mechanism is also very complex, moreover, the applied blood sample amount is often between 0.3 and 1.0 micrometer, therefore, present technical level can only bring a relatively accurate detection result. According to the statutory requirements of biosensor that is used to detect the glucose concentration of human blood from U.S. FDA: as long as the positive or negative deviation of over 95% of detection result and standard glucose concentration is within 20%, the product is considered as a qualified one. It can be seen that it is very difficult technically to improve the detection accuracy of the glucose concentration of human blood by biosensor.
According to the statistic data of International Diabetes Federation (IDF) in 2011, diabetes patients in the worldwide have been increased to 366 million at present, and this number is expected to reach nearly 600 million within 20 years. In China, the prevalence rate of diabetes has been increased by nearly two times within 10 years. In 2010, the prevalence rate of Chinese adult diabetes was 9.7%, and diabetes patients have been reached to more than 140,000,000. Due to the huge cardinal number, even if the testing accuracy rate can be increased by 1%, then, in the world, 3.6 million person-time will be avoid of being misdiagnosed, so there will be 3.6 million people benefit from that, which would bring considerable economic and social interests.
It is reminded by medical specialists that diabetes is an important part of non-infectious diseases. Lacking of reasonable monitor and treatment, the long-term hyperglycemia of diabetes patients may lead to damages of multisystem in the body and cause various acute and chronic complications, such as cardiovascular disease, diabetic nephropathy, diabetic neurogenic lesion and diabetic retinopathy lesion and so on. Therefore, the accuracy of testing is very important, for it can effectively make sure that both patients and doctors notice the changes of illness and revise the pharmacy dosage and eating habits as soon as possible, so as to prevent the patients from being a state of hyperglycemia and from horrible complication. Whether the reaction reagents have been correctly distributed at the electrode is a key factor that has always been ignored by people while ensures the accuracy of the whole testing results.